Vaccinia virus is a large DNA virus that replicates within the cytoplasm of eukaryotic cells. We have succeeded in using this virus as a eukaryotic cloning vector. The initial step was construction of plasmid recombinants containing foreign DNA flanked by vaccinia virus DNA sequences. The plasmid DNA was then introduced into cells infected with vaccinia virus where homologous recombination occurred. Selection of viral recombinants containing foreign DNA inserted into the vaccinia thymidin kinase gene was achieved by plaque assay in the presence of 5-bromodeoxyuridine. Alternatively, the herpes virus thymidine kinase gene was inserted into a non-essential region of the genome of thymidine kinase negative vaccinia virus mutants, and selection was achieved by plaque assay in the presence of methotrexate. Expression of the herpes virus gene was dependent on vaccinia virus regulatory sequences. Restriction endonuclease analysis, agarose gel electrophoresis, and hybridization to 32P-labeled DNA was used to confirm that site-specific recombination had occurred. The distinctive enzymatic properties of the herpes virus thymidine kinase was used to confirm expression of this gene.